scholarly journals HOPS drives vacuole fusion by binding the vacuolar SNARE complex and the Vam7 PX domain via two distinct sites

2011 ◽  
Vol 22 (14) ◽  
pp. 2601-2611 ◽  
Author(s):  
Lukas Krämer ◽  
Christian Ungermann
Keyword(s):  
Membrane Fusion ◽  
Snare Complex ◽  
Endomembrane System ◽  
Attachment Protein ◽  
Vacuole Fusion ◽  
Px Domain ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Membrane Tethering

Membrane fusion within the endomembrane system follows a defined order of events: membrane tethering, mediated by Rabs and tethers, assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes, and lipid bilayer mixing. Here we present evidence that the vacuolar HOPS tethering complex controls fusion through specific interactions with the vacuolar SNARE complex (consisting of Vam3, Vam7, Vti1, and Nyv1) and the N-terminal domains of Vam7 and Vam3. We show that homotypic fusion and protein sorting (HOPS) binds Vam7 via its subunits Vps16 and Vps18. In addition, we observed that Vps16, Vps18, and the Sec1/Munc18 protein Vps33, which is also part of the HOPS complex, bind to the Q-SNARE complex. In agreement with this observation, HOPS-stimulated fusion was inhibited if HOPS was preincubated with the minimal Q-SNARE complex. Importantly, artificial targeting of Vam7 without its PX domain to membranes rescued vacuole morphology in vivo, but resulted in a cytokinesis defect if the N-terminal domain of Vam3 was also removed. Our data thus support a model of HOPS-controlled membrane fusion by recognizing different elements of the SNARE complex.

scholarly journals Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1

2012 ◽  
Vol 23 (2) ◽  
pp. 337-346 ◽  
Author(s):  
Francesca Morgera ◽  
Margaret R. Sallah ◽  
Michelle L. Dubuke ◽  
Pallavi Gandhi ◽  
Daniel N. Brewer ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Secretory Pathway ◽  
Secretory Vesicle ◽  
Snare Complex ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Membrane Bound ◽  
Sm Protein ◽  
Snare Complex Formation

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function—it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6–Sec1 interaction is exclusive of Sec6–Sec9 but compatible with Sec6–exocyst assembly. In contrast, the Sec6–exocyst interaction is incompatible with Sec6–Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6–exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.

scholarly journals Reduced O-GlcNAcylation of SNAP-23 promotes cisplatin resistance by inducing exosome secretion in ovarian cancer

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Luomeng Qian ◽  
Xiaoshan Yang ◽  
Shaohui Li ◽  
Hang Zhao ◽  
Yunge Gao ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Protein Modification ◽  
Snare Complex ◽  
Ovarian Cancer Cells ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor

AbstractExosomes have been associated with chemoresistance in various cancers, but such a role in ovarian cancer is not yet clear. Here, using in vitro cell-based and in vivo mouse model experiments, we show that downregulation of O-GlcNAcylation, a key post-translational protein modification, promotes exosome secretion. This increases exosome-mediated efflux of cisplatin from cancer cells resulting in chemoresistance. Mechanistically, our data indicate that downregulation of O-GlcNAclation transferase (OGT) reduces O-GlcNAclation of SNAP-23. Notably, O-GlcNAcylation of SNAP-23 is vital for regulating exosome release in ovarian cancer cells. Reduced O-GlcNAclation of SNAP-23 subsequently promotes the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of SNAP-23, VAMP8, and Stx4 proteins. This enhances exosome release causing chemoresistance by increasing the efflux of intracellular cisplatin.

scholarly journals SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events

2017 ◽  
Vol 45 (6) ◽  
pp. 1271-1277 ◽  
Author(s):  
Kamilla M.E. Laidlaw ◽  
Rachel Livingstone ◽  
Mohammed Al-Tobi ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Membrane Fusion ◽  
Molecular Mechanisms ◽  
Membrane Receptors ◽  
Multivesicular Bodies ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Plasma Membrane Receptors ◽  
Regulated Trafficking ◽  
Receptor Family

Trafficking within eukaryotic cells is a complex and highly regulated process; events such as recycling of plasma membrane receptors, formation of multivesicular bodies, regulated release of hormones and delivery of proteins to membranes all require directionality and specificity. The underpinning processes, including cargo selection, membrane fusion, trafficking flow and timing, are controlled by a variety of molecular mechanisms and engage multiple families of lipids and proteins. Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. We will describe how these proteins are controlled in a range of regulated trafficking events, with particular emphasis on the insulin-stimulated delivery of glucose transporters to the surface of adipose and muscle cells. Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion.

scholarly journals A Vacuolar v–t-SNARE Complex, the Predominant Form In Vivo and on Isolated Vacuoles, Is Disassembled and Activated for Docking and Fusion

1998 ◽  
Vol 140 (1) ◽  
pp. 61-69 ◽  
Author(s):  
Christian Ungermann ◽  
Benjamin J. Nichols ◽  
Hugh R.B. Pelham ◽  
William Wickner
Keyword(s):  
Atp Hydrolysis ◽  
Complex Structure ◽  
Snare Complex ◽  
Functional Aspect ◽  
Independent Function ◽  
Attachment Protein ◽  
Vacuole Fusion ◽  
Predominant Form ◽  
Target Membrane

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [α-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v–t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v–t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v–t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, α-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.

scholarly journals SNARE complexes of different composition jointly mediate membrane fusion in Arabidopsis cytokinesis

2013 ◽  
Vol 24 (10) ◽  
pp. 1593-1601 ◽  
Author(s):  
Farid El Kasmi ◽  
Cornelia Krause ◽  
Ulrike Hiller ◽  
York-Dieter Stierhof ◽  
Ulrike Mayer ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Vesicles ◽  
Snare Complex ◽  
The Other ◽  
Division Plane ◽  
Daughter Cells ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Membrane Type ◽  
Cell Division Plane

Membrane fusion is mediated by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes. Although membrane fusion is required for separating daughter cells in eukaryotic cytokinesis, the SNARE complexes involved are not known. In plants, membrane vesicles targeted to the cell division plane fuse with one another to form the partitioning membrane, progressing from the center to the periphery of the cell. In Arabidopsis, the cytokinesis-specific Qa-SNARE KNOLLE interacts with two other Q-SNAREs, SNAP33 and novel plant-specific SNARE 11 (NPSN11), whose roles in cytokinesis are not clear. Here we show by coimmunoprecipitation that KNOLLE forms two SNARE complexes that differ in composition. One complex is modeled on the trimeric plasma membrane type of SNARE complex and includes, in addition to KNOLLE, the promiscuous Qb,c-SNARE SNAP33 and the R-SNARE vesicle-associated membrane protein (VAMP) 721,722, also involved in innate immunity. In contrast, the other KNOLLE-containing complex is tetrameric and includes Qb-SNARE NPSN11, Qc-SNARE SYP71, and VAMP721,722. Elimination of only one or the other type of KNOLLE complex by mutation, including the double mutant npsn11 syp71, causes a mild or no cytokinesis defect. In contrast, the two double mutants snap33 npsn11 and snap33 syp71 eliminate both types of KNOLLE complexes and display knolle-like cytokinesis defects. Thus the two distinct types of KNOLLE complexes appear to jointly mediate membrane fusion in Arabidopsis cytokinesis.

scholarly journals Localization, Dynamics, and Protein Interactions Reveal Distinct Roles for ER and Golgi SNAREs

1998 ◽  
Vol 141 (7) ◽  
pp. 1489-1502 ◽  
Author(s):  
Jesse C. Hay ◽  
Judith Klumperman ◽  
Viola Oorschot ◽  
Martin Steegmaier ◽  
Christin S. Kuo ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Protein Interactions ◽  
The Other ◽  
Snare Proteins ◽  
Attachment Protein ◽  
Golgi Transport ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Coated Membranes ◽  
Factor Attachment Protein Receptor

ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin, rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs). However, all of the SNAREs were found on both COPII- and COPI-coated membranes, indicating that similar SNARE machinery directs both vesicle pathways. rsec22b and rbet1 do not appear beyond the first Golgi cisterna, whereas syntaxin 5 and membrin penetrate deeply into the Golgi stacks. Temperature shifts reveal that membrin, rsec22b, rbet1, and syntaxin 5 are present together on membranes that rapidly recycle between peripheral and Golgi-centric locations. GOS-28, on the other hand, maintains a fixed localization in the Golgi. By immunoprecipitation analysis, syntaxin 5 exists in at least two major subcomplexes: one containing syntaxin 5 (34-kD isoform) and GOS-28, and another containing syntaxin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subcomplexes appear to involve direct interactions of each SNARE with syntaxin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusion interfaces: the fusion of ER-derived vesicles with VTCs, and the assembly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform, membrin, and GOS-28 may function in intraGolgi transport.

scholarly journals A Novel Site of Action for α-SNAP in the SNARE Conformational Cycle Controlling Membrane Fusion

2008 ◽  
Vol 19 (3) ◽  
pp. 776-784 ◽  
Author(s):  
Marcin Barszczewski ◽  
John J. Chua ◽  
Alexander Stein ◽  
Ulrike Winter ◽  
Rainer Heintzmann ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Site Of Action ◽  
Snare Motif ◽  
Liposome Fusion

Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of α-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of α-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of α-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of α-SNAP potently inhibits Ca2+-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an α-SNAP mutant defective in NSF activation is used. We conclude that α-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for α-SNAP in the SNARE cycle that drives exocytotic membrane fusion.

scholarly journals Syntaxin-17 delivers PINK1/parkin-dependent mitochondrial vesicles to the endolysosomal system

2016 ◽  
Vol 214 (3) ◽  
pp. 275-291 ◽  
Author(s):  
Gian-Luca McLelland ◽  
Sydney A. Lee ◽  
Heidi M. McBride ◽  
Edward A. Fon
Keyword(s):  
Common Ancestor ◽  
Vesicle Transport ◽  
Late Endosome ◽  
Snare Complex ◽  
Endomembrane System ◽  
Last Eukaryotic Common Ancestor ◽  
Transport Pathway ◽  
Attachment Protein ◽  
Mitochondrial Content ◽  
Protein Receptor

Mitochondria are considered autonomous organelles, physically separated from endocytic and biosynthetic pathways. However, recent work uncovered a PINK1/parkin-dependent vesicle transport pathway wherein oxidized or damaged mitochondrial content are selectively delivered to the late endosome/lysosome for degradation, providing evidence that mitochondria are indeed integrated within the endomembrane system. Given that mitochondria have not been shown to use canonical soluble NSF attachment protein receptor (SNARE) machinery for fusion, the mechanism by which mitochondrial-derived vesicles (MDVs) are targeted to the endosomal compartment has remained unclear. In this study, we identify syntaxin-17 as a core mitochondrial SNARE required for the delivery of stress-induced PINK1/parkin-dependent MDVs to the late endosome/lysosome. Syntaxin-17 remains associated with mature MDVs and forms a ternary SNARE complex with SNAP29 and VAMP7 to mediate MDV–endolysosome fusion in a manner dependent on the homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Syntaxin-17 can be traced to the last eukaryotic common ancestor, hinting that the removal of damaged mitochondrial content may represent one of the earliest vesicle transport routes in the cell.

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